Preparation of a universally usable, animal product free, defined medium for 2D and 3D culturing of normal and cancer cells

Since 1958, cell culture media supplemented with fetal bovine serum is used, despite the well-known concerns about animal welfare, reproducibility, reliability, relevance, and safety. To obliterate these concerns and increase scientific accuracy, we recently published an open access, publicly available paper on a defined medium composition to make it possible for any lab to prepare this medium. The medium supports routine culturing and cell banking as well as investigations of growth curves, dose response testing of compounds of cells in 2D and 3D, and cell migration; all important aspects for research and toxicology. Here we give a detailed description of how to mix the defined universal cell culture medium in 14 simple steps to support any entity that wishes to make it. We also list different normal and cancer cell lines that have been cultured in the defined medium.• Open source composition of animal product free universal cell culture medium• Protocols for mixing solutions of small xeno free molecules for supplementation• Protocols for mixing solutions of human proteins for supplementation

serum (FBS) and human platelet lysate (hPL), respectively.However, these supplements are undefined, characterized by batchto-batch variability, may induce a non-physiological, proliferating cellular phenotype, and bare the risk of contamination or even infections [5 , 6] .Together, this negatively affects safety, relevance, and reproducibility of research outcomes [7][8][9] .Especially, when culturing e.g.human cells in a non-human medium like FBS-supplemented medium, the transferability of the experimental results is questionable.Furthermore, it is an ethical necessity to replace animal-derived ingredients like FBS in the laboratory to maximize animal welfare [10][11][12][13] .
In 2023, Rafnsdóttir et al .published a paper on a defined, safe to use, animal free, and universally usable cell culture medium [14] .Table 1 lists the cell lines that were used in the published paper.Table 2 shows a list of different cancer and normal cell lines that since then have been adapted to the medium and that have been routine-cultured to ascertain stable growth.Presently we are performing experiments with HeLa, MCF-7, and MCF-10A cells (not published yet).Also, a number of dose response experiments have been performed with the pancreatic cancer cells lines (not published yet).
Here we provide a detailed protocol on how to prepare the defined medium.After the stock solutions of the components are prepared, the medium can be mixed by following only 14 simple steps.This protocol is using human-derived proteins, as well as human recombinant proteins in accordance with our publication Rafnsdóttir et al. [14] .However, it should be possible to use only recombinant proteins (e.g.recombinant human serum albumin [16] ) to even further increase reproducibility.
In addition, although the medium has been used for a variety of cell lines and we define it as universal, we hope this can be a starting point for abandoning the use of FBS and for further refinement to more exactly reproduce the human cellular environment.

Important information before preparation of the medium
Prior to the actual mixing of the defined medium, different phases have to be taken according to Fig. 1 .First, purchase all components needed for the medium.The company products displayed below are suggestions.Products with similar properties from different providers than listed here can be used for the preparation of the medium as well, e.g.human serum albumin from SeraCare1  (product number 1850-0028) or human placenta laminin [17] from THT Biomaterials2 (product number THT0201), instead of those listed below in Tables 4 and 5 .
The basis for our medium is DMEM/F12, which was developed for cells in monolayer culture and has a bicarbonate content for the use in an incubator with 5% CO 2 to obtain correct pH.Please observe that the original DMEM has a bicarbonate content that provides the correct pH when used in an incubator with 10% CO 2 .Thus, it should never be used in an incubator with 5% CO 2 , which unfortunately is found in publications and cell bank recommendations regarding medium for cell lines.
Additionally, the DMEM/F12 product mentioned here contains phenol red.Since phenol red is a xenobiotic with low estrogenic activity, a medium without phenol red would be preferred.Phenol red is added for visual pH control but with knowledge of how bicarbonate functions in pH control, phenol red can be omitted [14] .
Furthermore, this medium is made for cells that require attachment and has only been tested for such cells.We have not done any studies on cells in suspension culture with the exception of human CD4 + T-cells that were activated by CD3/CD28 microbeads to stimulate cell proliferation in the defined medium using RPMI1640 instead of DMEM/F12 (not published).When using DMEM/F12, the final concentration of each (non-protein) component in our defined medium can be seen in Table 26 .Our medium mimicks the complexity of human serum and industrial manufacturing will lower its production price significantly [24] .
Instead of coating the culture surfaces with fibronectin separately, we add fibronectin directly to the culture medium.Hence, when cells are seeded in the defined medium with fibronectin at passaging, the tissue culture surface will be covered with sufficient amounts of fibronectin.This also means that for the replacement medium in between passaging, use the medium without fibronectin as described in Rafnsdóttir et al. [14] .We have found that many cell lines do not appear to thrive when fibronectin is present in replacement medium.
Most cell culture laboratories already purchase concentrated solutions of cell culture components such as glutamine, sodium pyruvate, non-essential amino acid, and penicillin-streptomycin.We always aliquot these in 5 ml portions, which results in the desired concentration in 500 ml medium and keep the tubes frozen at − 20 °C.If possible, it is good to avoid antibiotics like penicillinstreptomycin as they also are xenobiotics.The half-life of antibiotics is quite short at 37 °C, about 2 days for penicillin and about 4 days for streptomycin.When sampling medium for mycoplasma testing, the cells should be cultured for 2 weeks in antibiotics free medium.This is a test of good sterile technique and possibly stimulates the constant use of medium without antibiotics.Some proteins needed for the defined medium are purchased as solutions and we suggest aliquoting as seen below.
T. Weber, J. Bajramovic and S. Oredsson MethodsX 12 (2024) 102592 When all solutions are prepared, thawed, and placed in the laminar air-flow (LAF) bench, the final mixing takes approximately 30 min for a person that is used to pipetting.Do not turn on the light in the LAF bench.As can be found in the protocols below, some components are light sensitive, and thus, the entire medium is light sensitive.The light sensitivity of media has been tested and reported and in general, all media are light sensitive [18 , 19] .Vitamins and retinoic acid mainly contribute to the light sensitivity [20][21][22] .We have not performed a systematic investigation of the light sensitivity of the defined medium but rely on published data.We have never had problems working in the LAF bench with the light off and have used the medium in courses with undergraduate students with no problems or complaints.
The complete medium is stored at 4 °C and we recommend that it is used within two months.Freezing the medium at − 20 °C will extend this approximately to four months; however, we have not performed a systematic storage time test of the complete defined medium a − 20 °C.Below you will find more information about aliquoting the mixed medium to minimize pH changes as well as the possibility of degradation of components with repeated warming of the medium before addition to cells.

Recommended tissue culture plastic
Most tissue culture plastic is made of polystyrene, although this material is naturally hydrophobic.It therefore poorly supports cell adhesion and cells that require attachment will die.Tissue culture plastic for cell culturing is oxygen plasma-treated resulting in the insertion of oxygen containing groups in the styrene molecules.In contact with water, ionization results in a negatively charged surface [23] .
Our experience shows that cells thrive better in the defined medium when cultured on Corning 3 Primaria tissue culture plastic [14] .The reason is that these plastics contain ammonium groups besides the oxygen containing groups and therefore become both positively and negatively charged in contact with water.

Mixing of the defined medium with prepared stock solutions
This is a description of how to combine the different components (see Tables 3-5 ) with the respective solvents (see Table 6 ).Enclosed are individual protocols for different components.The final mixing takes approximately 30 min for a person that is used to pipetting when all components are thawed and placed in the LAF bench.Do not turn on the light in the LAF bench.The medium is light sensitive.Please note that ergocalciferol, as mentioned in the original medium description [14] was found to be dispensable and can be dismissed.

Materials needed in the LAF bench
Automatic pipettes and tips to pipet different volumes from 10 μl to 250 μl.Pipettes 5 and 10 ml (preferably glass pipettes to reduce environmental impact).Sterile bottles for medium aliquoting and for collecting surplus DMEM/F12.Procedure 1. Remove 43.7 ml of the 500 ml DMEM/F12 (to compensate for added volumes).Save this surplus separately in a sterile flask.Mark well!You can collect medium in the same flask until you have enough to make more defined medium.2. Add 5 ml 200 mM L-glutamine.3. Add 5 ml 10 mM NEAA giving.4. Add 5 ml 100 mM sodium pyruvate.5. Add 5 ml penicillin-streptomycin (optional).If this is not used, re-add 5 ml of DMEM/F12.6. Add 2.5 ml transferrin solution.7. Add 100 μl insulin solution.
8. Turn off the light in the LAF bench.9. Thaw the Eppendorf tubes kept at − 80 °C in the dark until they reach room temperature.Spin at 2000 g for 30 s before pipetting to the medium.10.Add 20 ml of the HSA solution.Mix well.11.Now we usually divide the medium into two sterile 250 ml bottles.Label one bottle: "Defined medium without fibronectin for medium replacement " plus date.Caution: Fibronectin needs to be thawed slowly without disturbance at 37 °C to prevent clumping/precipitation. 12. Label the other bottle: "Defined medium with fibronectin for passaging " plus date.Add 250 μl fibronectin solution to this flask.
Keep the remaining fibronectin at 4 °C.It can be stored at 4 °C for 3 months.13.If you decide to make 500 ml defined medium with fibronectin, skip the steps 11 and 12 and add 500 μl of the fibronectin solution instead.14.Store at 4 °C for a maximum of 2 months or freeze at − 20 °C for 4 months.

Mixing of solutions with small organic and inorganic molecules
A number of proteins and chemicals are purchased as powders and below are protocols for making solutions.An important part of making solutions from powdery compounds is the possibility to weight with a high precision scale to reach a satisfactory accuracy and reproducibility.The scale we use has a precision of 0.00001 g.However, we recommend not weighing less than 100 μg .
Please note that before weighing laboratory materials stored below room temperature, allow these to come to room temperature.Otherwise, mass errors could occur due to condensation.
Be beware of the fact, that weighing small and precise masses of solids is more challenging than pipetting small and precise volumes of liquids.Therefore, to keep the concentration at the desired level, it might be easier to add the liquid amount according to the weighed solid mass.It is preferable to weigh slightly more than the exact amounts suggested below in the protocols, to not fall below the needed volume for the subsequent steps.
For instance: To receive a desired concentration of 0.2 μg/μl, you need to add precisely 1000 μg of solid Y to precisely 5000 μl of liquid X.If you have weighed 1094 μg of solid Y instead, you could receive the exact same desired concentration by adding 5470 μl of liquid X.

X = 5470 μl
Keep this in mind when preparing all compounds that need weighing.

How to prepare the stock solutions
Here you can find the protocols for the separate stock solutions.Once they are prepared, you just have to thaw and mix them according to the 14-step procedure mentioned above.An excel sheet for ticking off compounds while mixing the medium can be found in the supplementary materials.

Table 7
Preparation of the ethanol solution.

Product
Supplemented concentration Amount in the aliquot (25 μl) 4-Aminobenzoic acid 12 ng/ml 6.0 μg Cholesterol 50 ng/ml 25 μg Lipoic acid 50 ng/ml 25 μg Linoleic acid 1 μg/ml 500 μg General Information These compounds are soluble in gradient grade ethanol.Thus, start by preparing a stock solution for each compound for receiving the supplemented concentration in 500 ml defined medium as shown above.

Preparation Table 8
Preparation of the NaOH solution.

Product
Supplemented concentration Amount in the aliquot (50 μl) Folic acid 330 ng/ml 165 μg Uracil 75 ng/ml 37.5 μg Xanthine 85 ng/ml 42.5 μg General Information These compounds are soluble in NaOH.Thus, start by preparing a stock solution for each compound for receiving the supplemented concentration in 500 ml defined medium as shown above.Then combine these according the instructions.Preparation 1. Folic acid: Weigh 16.5 mg folic acid and dissolve in 500 μl 0.5 M NaOH, yielding a desired concentration of 33 μg/μl.You will use 500 μl in step 5. 2. Uracil: Weigh 3.75 mg uracil and dissolve in 500 μl 0.5 M NaOH, yielding a desired concentration of 7.5 μg/μl.You will use 500 μl in step 5.

General Information
These compounds are soluble in H 2 O. Thus, start by preparing a stock solution for each compound for receiving the supplemented concentration in 500 ml defined medium as shown above.Then combine these according the instructions.Some compounds are light sensitive.Work under subdued light.Preparation T. Weber, J. Bajramovic and S. Oredsson MethodsX 12 (2024) 102592

Table 10
Preparation of the all-trans retinoic acid solution.

Product
Supplemented concentration Amount in the aliquot (10 μl) All-trans -retinoic acid 25 ng/ml 12.5 μg General Information All-trans -retinoic acid is highly light sensitive and sensitive to air.Work in subdued light i.e. turn off the light in the LAF bench and dim the light in the room.Do not work with powder or concentrated all-trans -retinoic acid if you are pregnant.The compound is teratogenic.Dissolve all retinoic acid.Prepare a stock solution for receiving the supplemented concentration in 500 ml defined medium as shown above.Preparation 1. Open the glass ampoule with all-trans -retinoic acid with care.2. Dissolve all the powder (50 mg) in 2000 μl of 100 % DMSO (tissue culture grade), yielding a desired concentration of 25 μg/μl.You need to transfer to a sterile test tube.Make sure all powder is dissolved.3. Add 950 μl of DMSO to a sterile Eppendorf tube.4. Then add 50 μl of the concentrated solution of all-trans -retinoic acid. 5. Label sterile Eppendorf tubes with "Retinoic acid ".Mark the lid with RA.The solution made is for many tubes but we suggest preparing 50 tubes.6. Aliquot in the sterile Eppendorf tubes (10 μl per tube).7. Store at − 80 °C well protected from light for six months.

Note
All-trans -retinoic acid may not be required for some cell lines that can be induced to differentiate by retinoic acid treatment.However, the all-trans -retinoic acid concentration used here is very low (83.3 nM) compared to the concentrations used when inducing differentiation e.g. in SH-SY5Y neuroblastoma cells (1-10 μM).

Table 11
Preparation of the -estradiol solution.

Product
Supplemented concentration Amount in the aliquot (10 μl) -Estradiol 0.5 pg/ml 250 ng General Information Prepare a stock solution for receiving the supplemented concentration in 500 ml defined medium as shown above.Preparation

Table 12
Preparation of the hydrocortisone solution.

Product
Supplemented concentration Amount in the aliquot (20 μl) Hydrocortisone 0.25 ng/ml 125 ng General Information Prepare a stock solution for receiving the supplemented concentration in 500 ml defined medium as shown above.Preparation 1. Weigh 1 mg of hydrocortisone in a microcentrifuge tube and dissolve in 2000 μl 99.9 % ethanol, yielding a desired concentration of 0.5 μg/μl.

Table 13
Preparation of the triiodothyronine solution.

Fig. 1 .
Fig. 1.Schematic presentation of the phases of the medium preparation.

3 .
Xanthine: Weigh 4.25 mg xanthine and dissolve in 1000 μl 1 M NaOH, yielding a desired concentration of 4.25 μg/μl.You will use 1000 μl in step 5. 4. Add the volumes in bold to a new test tube.This will result in 2 ml solution.Add 3 ml of 0.5 M NaOH, resulting in a total volume of 5000 μl. 5. Sterile filter the solution.6. Label sterile Eppendorf tubes with "NaOH solution ".Mark the lid with a N.The solution made is for 100 tubes but we suggest preparing 50 tubes.7. Aliquot in the sterile Eppendorf tubes (50 μl per tube).8. Store at − 80 °C.
a stock solution for receiving the supplemented concentration in 500 ml defined medium as shown above.Preparation 1. Weigh 500 μg of triiodothyronine and add 1000 μl 100 % DMSO, yielding a desired concentration of 0.5 μg/μl.2. Add 490 μl 100 % DMSO to a sterile Eppendorf tube.3. Add 10 μl of the triiodothyronine solution to the tube with DMSO. 4. Label sterile Eppendorf tubes with Triiodothyronine.Mark the lid with a T. 5. Aliquot in the sterile Eppendorf tubes (10 μl per tube).6. Store at − 80 °C

Table 5
Components stored (aliquoted in Eppendorf tubes together in plastic bags) at − 80 °C.

Table 7 :
Preparation of the ethanol solution.

Table 10 :
Preparation of the all-trans retinoic acid solution.

Table 11 :
Preparation of the -estradiol solution.

Table 12 :
Preparation of the hydrocortisone solution.

Table 13 :
Preparation of the triiodothyronine solution.

Table 14 :
Preparation of the basic fibroblast growth factor.

Table 15 :
Preparation of the collagen solution.

Table 16 :
Preparation of the epidermal growth factor solution.

Table 20 :
Preparation of the insulin-like growth factor 1 solution.ThermoFisher Scientific a PHG0071 Platelet-derived growth factor AA (PDGF) See

Table 21 :
Preparation of the platelet-derived growth factor AA solution.

Table 6
Solvents needed for the preparation of the medium components.Stored at + 20 °C.

Table 9
Preparation of the H 2 O solution.